Cytochrome P4502E1 (CYP2E1) and inducible heme oxygenase 1 (HO-1) and a number of other proteins are bimodally targeted to mitochondria in addition to their well-established ER or cytoplasmic destination. Work in the PI's laboratory has led to the discovery of a new family of "chimeric" signals, which direct the bimodal targeting of CYPs, GSTs, and a number of other signaling molecules to more than one subcellular compartment. Mitochondria-targeted CYP2E1 and HO-1 induced oxidative stress, reduced activity of cytochrome c oxidase and other complexes and induced rho zero phenotypes in yeast. A pharmacogenetic screen of human liver bank showed inter-individual variations in mt-CYP2E1 levels which is likely to be associated with mutations that affect targeting efficiency of the protein. The objective of this application is to initiate new studies on the genetic basis for increased mitochondrial import of CYP2E1 and HO-1 and the role of these proteins in alcohol-mediated liver and heart injury using a multi-prong approach: 1) Investigate the mechanistic roles of mitochondrial CYP2E1 and HO-1 in ROS production, modulating the structure/function of cytochrome c oxidase and also other electron transfer complexes. Ethanol mediated ROS production and mt-dysfunction will be studied in stable cell lines expressing predominantly mt- or predominantly mc-targeted enzymes, and the effects of specific inhibitors of CYP2E1 and HO-1 in attenuating the damage will be tested. We will use a newly developed mitochondria-specific mito-DEPMPO EPR probe and also fluorescent probes for ROS measurement. Mitochondrial oxidative stress, mtDNA damage and functional parameters in cells treated with alcohol will be studied. In parallel yeast expression system will be used to assess cell toxicity. 2) Investigate the levels of mt-CYP2E1, and mt-HO-1 in the livers and hearts of human tissue bank from alcoholic and non-alcoholic patients. We will investigate the structure/function of CcO, mtDNA integrity and mt function in these patient samples. We will also analyze the 5'or 3'ends of mRNAs encoding the signal regions of the two proteins to identify human variants with altered mt-targeting efficiency to understand the genetic basis of inter-individual variations in mt contents of these proteins in the liver and heart tissues and to assess any correlation between mt-protein levels and alcohol mediated tissue injury. 3) Evaluate the ability of mitochondria-targeted antioxidant, Mito-Q, and inhibitors of CYP2E1, and HO-1 in attenuating alcohol-mediated lesions in two metabolically distinct tissues, the liver and heart. We will study hepatic lesions, mt-dysfunction, myocardial ischemia/reperfusion injury in alcohol-treated or alcohol + LPS treated rats. Another objective is to develop molecular interventions in the form of mt-targeted peptides that are designed to interfere with the catalytic activities of mt-CYP2E1, HO-1 with a view to reduce or prevent alcoholic tissue damage. Myocardial ischemia/reperfusion will be carried out using Langendorff perfusion system. These results should provide valuable new insights on genetic basis of alcohol induced mitochondrial toxicity and tissue damage, in addition to help identifying important molecular targets for developing therapeutic interventions. The objective of this application is to initiate studies on the role of bimodally targeted CYP2E1 and HO-1 in alcohol induced toxicity to the liver and heart using cell and animal models. Human genetic variant forms of CYP2E1 and HO-1 with vastly increased mitochondrial targeting will be investigated to understand the genetic basis for inter-individual variations in alcohol induced toxicity. The proposed research project is based on the hypothesis that mitochondrially targeted CYP2E1 and HO-1 contribute to ROS production, oxidative stress and mitochondrial dysfunction as important factors leading to tissue injury.